U.S. patent application number 16/000709 was filed with the patent office on 2019-03-07 for antimicrobial peptides and methods of use thereof.
The applicant listed for this patent is Riptide Bioscience, Inc.. Invention is credited to L. Edward Clemens, Jesse Jaynes, Henry W. Lopez, George R. Martin, Kathryn Woodburn.
Application Number | 20190071470 16/000709 |
Document ID | / |
Family ID | 56978879 |
Filed Date | 2019-03-07 |
![](/patent/app/20190071470/US20190071470A1-20190307-D00001.png)
United States Patent
Application |
20190071470 |
Kind Code |
A1 |
Jaynes; Jesse ; et
al. |
March 7, 2019 |
ANTIMICROBIAL PEPTIDES AND METHODS OF USE THEREOF
Abstract
Aspects of the present invention relate to peptides having
antimicrobial activity. In certain aspects, the invention relates
to peptides having potent antimicrobial activity, broad-spectrum
antimicrobial activity, and/or the ability to kill otherwise
antibiotic-resistant microbes, or microbes protected by
biofilms.
Inventors: |
Jaynes; Jesse; (Auburn,
CA) ; Clemens; L. Edward; (Sacramento, CA) ;
Lopez; Henry W.; (Napa, CA) ; Martin; George R.;
(Rockville, MD) ; Woodburn; Kathryn; (Saratoga,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Riptide Bioscience, Inc. |
Vallejo |
CA |
US |
|
|
Family ID: |
56978879 |
Appl. No.: |
16/000709 |
Filed: |
June 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15078794 |
Mar 23, 2016 |
10017542 |
|
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16000709 |
|
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62137206 |
Mar 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/00 20130101;
A61P 33/02 20180101; A61K 38/00 20130101; A61P 31/10 20180101; C07K
7/06 20130101; A61K 45/06 20130101; C07K 7/08 20130101; A61P 31/04
20180101 |
International
Class: |
C07K 14/00 20060101
C07K014/00; C07K 7/08 20060101 C07K007/08; C07K 7/06 20060101
C07K007/06; A61K 45/06 20060101 A61K045/06 |
Claims
1. An antimicrobial composition comprising an antimicrobial
peptide, wherein the antimicrobial peptide is 35 amino acid
residues or less in length and comprises an amphipathic region
having an amino acid sequence according to Formula 1 or 2:
TABLE-US-00018 XYYXXYYXXYXXYYXXYY (Formula 1) YYXXYYXXYXXYYXXYYX
(Formula 2)
wherein: each Y is a hydrophobic residue; each X is independently a
hydrophilic amino acid residue.
2. The antimicrobial composition of claim 1, wherein the
antimicrobial peptide is effective in killing at least one
bacterial, fungal, or protozoan organism.
3.-5. (canceled)
6. The antimicrobial composition of claim 1, wherein the
antimicrobial peptide comprises an amphipathic region having a
cationic surface that binds to the membrane of a microbial
pathogen.
7. The antimicrobial composition of claim 1, wherein the
antimicrobial peptide comprises an amino acid sequence selected
from RP-443 and RP-444 (SEQ ID NOs: 22-23).
8. The antimicrobial composition of claim 1, wherein the
antimicrobial peptide further comprises a hydrophobic tail region
on the N-terminus, C-terminus, or both, wherein the hydrophobic
tail region has a sequence of from 4 to 10 hydrophobic amino
acids.
9. The antimicrobial composition of claim 8, wherein the
hydrophobic tail region comprises the amino acid sequence set forth
in SEQ ID NO: 4.
10. An antimicrobial composition comprising an antimicrobial
peptide, wherein the antimicrobial peptide is 35 amino acid
residues or less in length and comprises two amphipathic regions
joined together by a linker comprising a bubble region of Formula
5: TABLE-US-00019 C(Y/X)(Y/X)(Y/X)(Y/X)C
wherein: each Y is a hydrophobic residue; each X is independently a
hydrophilic amino acid residue.
11. The antimicrobial composition of claim 10, wherein the two
amphipathic regions are each 5 or more residues in length and
consist of an alternating sequence of one hydrophobic (Y) amino
acid residue and one hydrophilic amino acid residue (X).
12. The antimicrobial composition of claim 11, wherein the bubble
region comprises the sequence CLGRFC (SEQ ID NO: 7).
13. (canceled)
14. The antimicrobial composition of claim 10, wherein the two
amphipathic regions have the same amino acid sequence.
15. The antimicrobial composition of claim 10, wherein the two
amphipathic regions have different amino acid sequences.
16. The antimicrobial composition of claim 10, wherein the
antimicrobial peptide comprises an amino acid sequence RP-504 (SEQ
ID NO: 27).
17. A pharmaceutical composition comprising the antimicrobial
composition of claim 1 and a pharmaceutically acceptable
carrier.
18. (canceled)
19. The pharmaceutical composition of claim 17, wherein the
composition is formulated for oral administration and further
comprises an enteric coating.
20. The pharmaceutical composition of claim 17, wherein the
composition is formulated for topical delivery in a form selected
from a gel suspension, a cream, microneedle, and infused into a
bandage or topical patch.
21. The pharmaceutical composition of claim 17, further comprising
an additional bioactive agent.
22. The pharmaceutical composition of claim 21, wherein the
additional bioactive agent is selected from an antimicrobial agent,
an anti-inflammatory drug, an anti-nausea drug, an anti-pain
medication, and combinations thereof.
23. The pharmaceutical composition of claim 17, wherein the
composition is formulated to be coated on the surface of an
implantable medical device.
24. (canceled)
25. A method of treating or preventing a microbial infection in a
subject in need thereof, the method comprising administering a
pharmaceutical composition according to claim 1 to the subject.
26. The method of claim 25, wherein the pharmaceutical composition
is administered to the subject orally, parenterally, or
topically.
27. The method of claim 25, wherein the pharmaceutical composition
is administered to the subject by applying the composition to a
surface of a medical device prior to inserting the medical device
into the subject.
28. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Pursuant to 35 U.S.C. .sctn. 119 (e), this application
claims the benefit of priority to U.S. Provisional Patent
Application Ser. No. 62/137,206, filed Mar. 23, 2015, the
disclosure of which application is hereby incorporated by reference
herein in its entirety.
FIELD OF THE INVENTION
[0002] The invention generally relates to peptides having
antimicrobial activity. More particularly, the invention relates to
peptides having potent antimicrobial activity, broad spectrum
anti-bacterial activity, and/or the ability to kill otherwise
antibiotic-resistant bacteria, or bacteria protected by
biofilms.
BACKGROUND OF THE INVENTION
[0003] Antibiotic resistance is a major health problem. In part
this is attributed to the widespread use of antibiotics not only in
medicine, but in agriculture and animal husbandry. Such overuse,
while killing susceptible organisms, has also created a powerful
selection bias toward antibiotic resistant bacteria. The resulting
strains pose a particular problem for individuals with weakened
immune systems. In addition, they represent an increasingly serious
problem for patients in hospitals.
[0004] In addition to exhibiting inherited antibiotic resistance,
many emerging bacterial strains can exist in complex associations
known as biofilm. The structure of the biofilm constitutes a
physical barrier to antibiotic exposure. Biofilms can form in and
on tissues, particularly on chronic wounds and medical implants,
such as indwelling catheters, artificial organs, and the like,
where they have the potential to cause systemic infections
requiring heroic treatments. There is an urgent need for materials
that are active against antibiotic resistant organisms in both free
and biofilm form.
[0005] As part of their natural defense against bacteria, many
organisms, including insects, amphibians, mammals, and humans,
produce antimicrobial peptides. Such peptides are chemically
diverse. Some appear to act by penetrating the bacterial cell
membrane and destroying it. Others affect bacterial cellular
processes. Considerable selectivity is observed, with many of the
peptides targeting bacteria in preference to host cells.
Unfortunately, host produced antimicrobial peptides are not capable
of effectively eliminating a wide range of microbial agents,
including many antibiotic resistant bacterial strains.
Antimicrobial peptides capable of augmenting the host's
antimicrobial defenses are therefore desirable.
SUMMARY OF THE INVENTION
[0006] In certain embodiments, the invention provides antimicrobial
peptides. The peptides can have anti-bacterial, anti-fungal, and/or
anti-protozoal activity. The peptides can have the ability to kill
microbial strains that are resistant to conventional antibiotics.
In certain embodiments, antimicrobial peptides of the invention are
capable of killing microbes (e.g., bacteria) growing as a microbial
biofilm.
[0007] In certain embodiments, antimicrobial peptides of the
invention have amphipathic structure coupled with an overall
cationic charge, hydrophobicity, volume, and mass that generates
affinity for specific microbial membrane regions. Thus, the
antimicrobial peptides can include at least one amphipathic region.
The amphipathic region can include a cationic surface suitable for
binding to a bacterial membrane. The antimicrobial peptides can
include two amphipathic regions linked together by a third region,
such as a bubble region or a beta-turn. In certain embodiments, the
two amphipathic regions can dimerize, either as a heterodimer or
homodimer. Exchange of various domains from one peptide to another
or duplication of domains in the peptides is envisioned in the
invention to enhance activity, pharmacodynamics or similar features
important to clinical application.
[0008] The antimicrobial peptides of the invention comprise amino
acid residues. In certain embodiments, the amino acid residues are
naturally occurring L-amino acid residues. In some embodiments, one
or more amino acid residues in an antimicrobial peptide may be a
non-naturally occurring amino acid residue, a D-amino acid residue,
and/or a beta amino acid residue. In certain embodiments,
antimicrobial peptides of the invention have a sequence that is
resistant to proteolysis. For example, the peptides can include
amino acid residues, either naturally or non-naturally occurring,
that confer protease resistance.
[0009] In certain embodiments, the antimicrobial peptides have
specificity for non-mammalian cells. For example, the peptides can
be 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5 times, or more effective
at killing target microbial cells as compared to killing host
mammalian (e.g., human) cells. Some antimicrobial peptides of the
invention are active on one type of microbial organism but not on
others, thus providing antimicrobial selectivity. For example,
certain peptides of the invention can kill antibiotic-resistant
target bacteria while having minimal impact on other strains of
bacteria, particularly symbiotic bacteria (e.g., bacteria that
normally reside in the lumen of the gut of a mammal, such as a
human).
[0010] In some embodiments, the invention provides compositions,
particularly pharmaceutical compositions, which include one or more
antimicrobial peptides of the invention. Such compositions can be
formulated for oral administration, parenteral administration,
topical administration, or the like. Compositions formulated for
oral delivery can, for example, include an enteric coat, to ensure
that antimicrobial peptides contained therein reach the intestine
and beyond. Compositions formulated for topical delivery can be,
for example, suspended in a gel or creme or infused into a bandage,
to extend the duration of action of the antimicrobial peptides
contained therein. Alternatively, the antimicrobial peptides of the
invention can be coated on the surface of medical devices, such as
surgical instruments and indwelling medical devices (e.g.,
pacemakers, catheters, artificial joints, and the like), as a means
of preventing infection.
[0011] In some embodiments, the invention provides methods of
treating microbial infections, or prophylactically preventing such
infections. The methods can include administering a composition
containing one or more antimicrobial peptides of the invention. The
compositions can be administered orally, parenterally, topically,
or the like. Oral or parenteral administration can be used to
treat, for example, systemic infections. Topical administration can
be used to treat, for example, wounds or burns. For treatment of
patients that require an indwelling medical device, such as a
catheter or artificial joint, the treatment can include applying
one or more antimicrobial peptides to the medical device prior to
inserting the medical device into the patient. The methods can be
used to treat any of a wide range of animals, particularly mammals,
such as human, domesticated animals, farm animals, zoo animals,
wild animals, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a diagram of a hairpin peptide according to one
aspect of the invention.
[0013] FIG. 2 shows data for the activity of two peptides of the
present invention (designated "RP-439" and "RP-442"; SEQ ID NOs: 18
and 21, respectively) against select microbial organisms associated
with hospital-acquired infections as compared to the antibiotic
vancomycin. The organisms associated with hospital-acquired
infections tested were Staphylococcus aureus, Pseudomonas
aeruginosa, and Clostridium difficile.
DETAILED DESCRIPTION OF THE INVENTION
[0014] As discussed above, the invention disclosed herein relates
to antimicrobial polypeptides and methods of administering such
antimicrobial polypeptides to a subject to prevent or treat a
microbial infection.
[0015] Before the present invention is described in greater detail,
it is to be understood that this invention is not limited to
particular embodiments described, which as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0016] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0017] Certain ranges are presented herein with numerical values
being preceded by the term "about." The term "about" is used herein
to provide literal support for the exact number that it precedes,
as well as a number that is near to or approximately the number
that the term precedes. In determining whether a number is near to
or approximately a specifically recited number, the near or
approximating unrecited number may be a number which, in the
context in which it is presented, provides the substantial
equivalent of the specifically recited number.
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0019] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0020] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0021] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0022] The terms "peptide" and "polypeptide" are used synonymously
herein to refer to polymers constructed from amino acid
residues.
[0023] The term "amino acid residue," as used herein, refers to any
naturally occurring amino acid (L or D form), non-naturally
occurring amino acid, or amino acid mimetic (such as a peptoid
monomer).
[0024] The "length" of a polypeptide is the number of amino acid
residues linked end-to-end that constitute the polypeptide,
excluding any non-peptide linkers and/or modifications that the
polypeptide may contain.
[0025] A "linker" or "linker sequence" can be any moiety that links
two peptide sequences together. In some embodiments, a linker is an
amino acid sequence that is co-linear with the peptide sequences
being linked together, whereas in other embodiments a linker is a
separate moiety that is attached to the two peptide sequences,
e.g., via a covalent linkage. Linkers can be amino acid sequences
or be non-amino acid moieties. In certain embodiments, a linker is
used to facilitate dimerization of two amphipathic regions.
[0026] Hydrophobic amino acid residues are characterized by a
functional group ("side chain") that has predominantly non-polar
chemical properties. Such hydrophobic amino acid residues can be
naturally occurring (L or D form) or non-naturally occurring.
Alternatively, hydrophobic amino acid residues can be amino acid
mimetics characterized by a functional group ("side chain") that
has predominantly non-polar chemical properties. Conversely,
hydrophilic amino acid residues are characterized by a functional
group ("side chain") that has predominantly polar (charged or
uncharged) chemical properties. Such hydrophilic amino acid
residues can be naturally occurring (L or D form) or non-naturally
occurring. Alternatively, hydrophilic amino acid residues can be
amino acid mimetics characterized by a functional group ("side
chain") that has predominantly polar (charged or uncharged)
chemical properties. Examples of hydrophilic and hydrophobic amino
acid residues are shown in Table 1, below. Suitable non-naturally
occurring amino acid residues and amino acid mimetics are known in
the art. See, e.g., Liang et al. (2013), "An Index for
Characterization of Natural and Non-Natural Amino Acids for
Peptidomimetics," PLoS ONE 8(7):e67844.
[0027] Although most amino acid residues can be considered as
either hydrophobic or hydrophilic, a few, depending on their
context, can behave as either hydrophobic or hydrophilic. For
example, due to their relatively weak non-polar characteristics,
glycine, proline, and/or cysteine can sometimes function as
hydrophilic amino acid residues. Conversely, due to their bulky,
slightly hydrophobic side chains, histidine and arginine can
sometimes function as hydrophobic amino acid residues.
TABLE-US-00001 TABLE 1 Hydrophobic and Hydrophilic Amino Acid
Residues Hydrophilic Residues Hydrophobic Residues (X) (Y) Arginine
Tryptophan Histidine Phenylalanine Lysine Tyrosine Aspartic Acid
Isoleucine Glutamic Acid Leucine Asparagine Valine Glutamine
Methionine Pyrrolysine Cysteine Ornithine Threonine Serine Alanine
Proline Glycine Selenocysteine N-formylmethionine Norleucine
Norvaline
[0028] As described in further detail below, aspects of the present
disclosure include antimicrobial peptides having at least one
amphipathic region having a specific degree of cationic charge. In
certain embodiments, the antimicrobial peptide includes a tail
region (e.g., a hydrophobic tail sequence). In certain embodiments,
an antimicrobial peptide (or peptide agent) includes two or more
amphipathic regions. In such embodiments, two amphipathic regions
of an antimicrobial peptide (or peptide agent) are in the form of a
dimer, where the two amphipathic regions can have the same or
different amino acid sequences (i.e., be homodimer or a
heterodimer). In certain embodiments, the two (or more) amphipathic
regions are connected via a linker. The linker can be a contiguous
(or in-line) amino acid sequence or a non-amino acid moiety as
desired by a user. The linker can be, e.g., a bubble region or a
beta-turn region. In certain embodiments, the antimicrobial peptide
includes a polyproline helix structure.
[0029] Exemplary antimicrobial peptide sequences are shown below.
Additional antimicrobial peptides could be readily designed by one
skilled in the art by combining different regions of the exemplary
antimicrobial peptides in different ways as described herein.
Amphipathic Region
[0030] By amphipathic region is meant a peptide region that
possesses both hydrophobic and hydrophilic elements or
characteristics, for example, a peptide region possessing a
hydrophilic surface and a hydrophobic surface. A peptide region is
said to be in an amphipathic conformation when it exhibits an
amphipathic characteristic, which is often dependent on the
conditions under which the peptide was made and/or to which it has
been subjected. To be considered amphipathic, a peptide sequence
(or portion thereof) need not be in the amphipathic conformation at
all times. Rather, it is sufficient that the amphipathic
conformation be present at least 50%, 60%, 70%, 80%, or more of the
time.
[0031] In certain embodiments, an amphipathic region of an
antimicrobial peptide of the invention can be from 5 to 35 amino
acid residues in length, with at least 25% (e.g., 30%, 40%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, or more) of the amino acid sequence
of the amphipathic region exhibiting amphipathy. In certain
embodiments, an amphipathic region can include an alternating
sequence of 1 to 3 hydrophobic and 1 to 3 hydrophilic amino acid
residues. An amphipathic region can thus be represented by the
formula (X.sub.1-3Y.sub.1-3).sub.n, where X signifies a hydrophilic
amino acid residue, Y signifies a hydrophobic amino acid residue,
and n is an integer from 2 to 15. For example, an amphipathic
region can have a sequence according to Formula 1, Formula 2 (the
reverse of Formula 1), or Formula 3:
TABLE-US-00002 Formula 1: XYYXXYYXXYXXYYXXYY Formula 2:
YYXXYYXXYXXYYXXYYX Formula 3: XYXYXYXYXYXYXYX
[0032] Each hydrophobic amino acid residue Y is selected from the
group consisting of a naturally occurring hydrophobic amino acid, a
non-naturally occurring hydrophobic amino acid, and a hydrophobic
amino acid mimetic. Each hydrophilic amino acid residue X is
selected from the group consisting of a naturally occurring
hydrophilic amino acid, a non-naturally occurring hydrophilic amino
acid, and a hydrophilic amino acid mimetic. Often, the amphipathic
conformation will be associated with a particular secondary
structure, such as a helical structure. Thus, the amphipathic
region of an antimicrobial polypeptide can have an amphipathic
3.sub.10-helical conformation, an amphipathic .alpha.-helical
conformation, an amphipathic .pi.-helical conformation, or an
amphipathic polyproline helical conformation. Alternatively, the
amphipathic region of an antimicrobial polypeptide can have an
amphipathic .beta.-strand conformation.
[0033] In certain embodiments, the amphipathic region of an
antimicrobial peptide according to aspects of the present
disclosure includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10
or more) large hydrophobic amino acid residues. Examples of large
hydrophobic amino acid residues include tryptophan, phenylalanine,
and tyrosine. In addition, under certain circumstances, histidine
or arginine can be considered a large hydrophobic amino acid
residue. In certain embodiments, the amphipathic region of an
antimicrobial peptide according to aspects of the present
disclosure includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10
or more) small hydrophobic amino acid residues. Examples of small
hydrophobic residues include glycine, alanine, serine, cysteine,
valine, threonine, and proline. In certain embodiments, the
antimicrobial polypeptide has an amphipathic region that includes a
combination of large and small hydrophobic residues.
[0034] Specific examples of amphipathic regions include:
TABLE-US-00003 (SEQ ID NO: 1) RVFKKAFRKFKKLFKRAF; (SEQ ID NO: 2)
FARKFLKKFKRFAKKFVR; and (SEQ ID NO: 3) FKRKIKAKLRFKAKVRLK.
Cationic Charge/Surface
[0035] Antimicrobial polypeptides according to aspects of the
present disclosure include an amphipathic region having a cationic
surface. In certain embodiments, the amphipathic region has a
cationic charge (i.e., charge >0, e.g., +1, +2, +3, +4, +5, +6,
+7, +8, +9, +10 or more). Thus, in certain embodiments, an
amphipathic region of the disclosed peptides contains one or more
polar cationic amino acid residues (i.e., having positively charged
side chains). Examples of amino acid residues having positively
charged side groups (assuming physiological conditions) includes
lysine, typically arginine, and sometimes histidine. Accordingly,
an antimicrobial polypeptide can have an amphipathic region that
includes from 1 to 20 cationic amino acid residues. Thus, an
antimicrobial peptide of the invention can include polar amino acid
residues, at least 40% (e.g., 50%, 60%, 70%, 80%, 90%, or 100%) of
which are cationically charged (e.g., Arg, Lys, His).
Tail Region
[0036] In certain embodiments, an antimicrobial peptide includes a
tail region. A tail region of an antimicrobial peptide of the
invention can be from 3 to 15 amino acid residues in length, with
at least 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, or more) of the
amino acid residues in the tail region being hydrophobic. The tail
region can be located at either the N-terminus, the C-terminus, or
both termini of the antimicrobial peptide. In certain embodiments,
the tail region includes one polar amino acid for every 6 amino
acids. An example of a tail region sequence is shown in Formula 4,
where Y signifies hydrophobic amino acid residues.
TABLE-US-00004 Formula 4: YYYYY
[0037] Specific examples of antimicrobial peptides according to
aspects of the invention that include a tail region having the
sequence FAFAF (SEQ ID NO: 4) include (the tail region is
underlined):
TABLE-US-00005 (SEQ ID NO: 5) FAFAFRVFKKAFRKFKKLFKRAF; and (SEQ ID
NO: 6) FARKFLKKFKRFAKKFVRFAFAF.
Bubble Region
[0038] In certain embodiments, an antimicrobial peptide includes a
bubble region. A "bubble" region of an antimicrobial peptide of the
invention consists of a stretch of amino acid residues flanked by a
Cysteine residue (C) at each end (see FIG. 1). The stretch of amino
acid residues between the cysteine residues can be from 2 to 10
amino acid residues in length, and can be composed of any
combination of hydrophobic and hydrophilic amino acids. Bubble
regions can link two amphipathic regions and contribute to the
formation of a hairpin secondary structure by the antimicrobial
peptide (see FIG. 1). This region can thus be classified as a type
of "linker region" (as can other regions, described elsewhere
herein). In certain embodiments, the hairpin secondary structure
can significantly enhance antimicrobial activity.
[0039] A bubble region can have, for example, a sequence as shown
in Formula 5, where Y signifies hydrophobic amino acid residues and
X signifies hydrophilic amino acid residues.
TABLE-US-00006 Formula 5: C(Y/X)(Y/X)(Y/X)(Y/X)C
[0040] Specific examples of antimicrobial peptides of the invention
that include a bubble region having the sequence CLGRFC (SEQ ID NO:
7) include:
TABLE-US-00007 (SEQ ID NO: 8) KIRAKLCLGRFCIRAKLR; and (SEQ ID NO:
9) KIKARLCLGKFCIKARLK.
Dimerization
[0041] Without intending to be limited by theory, it is believed
that efficacy of the antimicrobial peptides of the invention
depends, in large part, on peptide dimerization and clustering on
the cell membrane of the target microbe (e.g., bacterial cell). It
is believed that dimers are more efficient at penetrating and,
ultimately, lysing the cell membrane. The formation of such dimers
can be thermodynamically more favorable when the peptides are
physically linked together, e.g., using linker regions. Linker
regions can include additional amino acid residues (e.g., like the
bubble region described above) or be non-amino acid-containing
linker moieties.
Beta Turn Region
[0042] A .beta.-turn sequence can be used to physically link
individual monomers, making intra-molecular interactions more
likely to take place. This appears to be particularly important for
amphipathic lytic peptides, as it allows their hydrophobic surfaces
to be protected from the aqueous phase. The .beta.-turn sequence
allows for two intra-chain amphipathic regions to form a dimer in
an antiparallel orientation. This region can thus be classified as
a type of "linker region" (as can other regions, described
elsewhere herein). For example, a monomer of SEQ ID NO: 6 (shown
above) killed greater than 2 logs of Staphylococcus aureus at a
concentration of 1.26 .mu.M, while the dimer of SEQ ID NO: 12
(shown below) killed greater than 2 logs of the same bacterium at a
concentration less than 0.156 .mu.M. (See Example 5).
[0043] A .beta.-turn sequence can be any .beta.-turn sequence known
in the art. A .beta.-turn sequence can have, for example, a
sequence as shown in SEQ ID NO: 10, where Y signifies hydrophobic
amino acid residues and X signifies hydrophilic amino acid residues
(i.e., any amino acid residue).
TABLE-US-00008 (SEQ ID NO: 10) (Y/X)GPGR(Y/X)
[0044] Specific examples of antimicrobial peptides of the invention
that include a .beta.-turn sequence having the sequence FGPGRF (SEQ
ID NO: 11) include:
TABLE-US-00009 (SEQ ID NO: 12)
FAFAFKAFKKAFKKFKKAFKKAFGPGRFAKKFAKKFKKFAKKFAKFAFAF
Polyproline Helix Secondary Structure
[0045] Without intending to be limited by theory, it is believed
that a helical structure in which a proline residue is repeated,
resulting in approximately 3.0 amino acid residues per turn rather
than the more normal 3.6 amino acid residues per turn, can result
in extended half-life for the resulting peptide. Such helices can
be formed, while maintaining the necessary structural features set
forth above, including an amphipathic region, cationic charge, and
optionally a tail region.
Examples of Antimicrobial Peptides
[0046] Examples of antimicrobial peptides according to aspects of
the invention are provided below in Table 2. These examples are
representative, and not meant to be limiting to the scope of the
invention. The "O" residues in the sequences listed below represent
the amino acid ornithine.
TABLE-US-00010 TABLE 2 Examples of antimicrobial peptides RP # SEQ
ID Amino Acid Sequence Na SEQ ID NO: 1 RVFKKAFRKFKKLFKRAF Na SEQ ID
NO: 2 FARKFLKKFKRFAKKFVR Na SEQ ID NO: 3 FKRKIKAKLRFKAKVRLK Na SEQ
ID NO: 5 FAFAFRVFKKAFRKFKKLFKRAF Na SEQ ID NO: 6
FARKFLKKFKRFAKKFVRFAFAF Na SEQ ID NO: 8 KIRAKLCLGRFCIRAKLR Na SEQ
ID NO: 9 KIKARLCLGKFCIKARLK RP-433 SEQ ID NO: 12
FAFAFKAFKKAFKKFKKAFKKAFGPGR FAKKFAKKFKKFAKKFAKFAFAF RP-434 SEQ ID
NO: 13 FAKKFAKKFKKFAKKFAKFAFAFGPGR FAFAFKAFKKAFKKFKKAFKKAF RP-435
SEQ ID NO: 14 MGFKLRAKIKVRLRAKIKL RP-436 SEQ ID NO: 15 CVOLFPVOLFPC
RP-437 SEQ ID NO: 16 CKLRFRGPGRIKVRLC RP-438 SEQ ID NO: 17
CPGFAKKFAKKFKKFAKKFAKFAFAF RP-439 SEQ ID NO: 18 KIRAKLCLGRFCIRAKLR
RP-440 SEQ ID NO: 19 KKKPKPPYLPKPKPPPFFPPKLPPKI RP-441 SEQ ID NO:
20 FAFAFKAFKKAFKKFKKAFKKAFGPC RP-442 SEQ ID NO: 21
FAFAFAFKKAFKKFKKAFKKAF RP-443 SEQ ID NO: 22 FAFAFOAFOOAFOOFOOAFOOAF
RP-444 SEQ ID NO: 23 FAOOFAOOFOOFAOOFAOFAFAF RP-445 SEQ ID NO: 24
FAKKFAKKFKKFAKKFAFAFAF RP-500 SEQ ID NO: 25
RLARIVGGFAOOFAOOFOOFAOOFAOF AFAF RP-501 SEQ ID NO: 26
CRLARIVCGGFAOOFAOOFOOFAOOFA OFAFAF RP-504 SEQ ID NO: 27
FOIOAOLGGCLGOFCGGIOAOLOF RP-505 SEQ ID NO: 28
OLOSLLKTLSOAOOOOLOTOOOAISO RP-507 SEQ ID NO: 29
ALWMTLOOOVLOAOAOALNAVLVGANA RP-508 SEQ ID NO: 30
AFAFTAOOOFAOFOAOFANFAFAGFNA
[0047] Accordingly, the invention further provides polypeptides
that include an amino acid sequence that is least 50% identical
(e.g., at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or more
identical) to any one of the antimicrobial polypeptides disclosed
herein (e.g., as shown in Table xx) and still retain at least one
antimicrobial property. In certain embodiment, such polypeptide
sequences include an amphipathic region having a cationic charge as
described in detail above. Moreover, such polypeptides may include
additional structural features as described herein, including: a
bubble region, a beta-turn region, a polyproline helix structure, a
tail, amphipathic region dimer, etc.
[0048] As such, in certain embodiments, the invention provides
polypeptides that include an amino acid sequence having from 1 to
10 amino acid differences (e.g., 10 or fewer, 9 or fewer, 8 or
fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer,
2 or fewer, or 1 amino acid difference) to any one of the
antimicrobial polypeptides disclosed herein (e.g., as shown in
Table xx) and still retain at least one antimicrobial property. An
"amino acid difference" as used herein includes: an amino acid
substitution, an amino acid insertion, a terminal amino acid
addition, an amino acid deletion, a terminal amino acid truncation,
or any combination thereof. A substituted amino acid residue (or
residues) can be unrelated to the amino acid residue being replaced
(e.g., unrelated in terms or hydrophobicity/hydrophilicity, size,
charge, polarity, etc.), or the substituted amino acid residue can
constitute similar, conservative, or highly conservative amino acid
substitution. As used herein, "similar," "conservative," and
"highly conservative" amino acid substitutions are defined as shown
in Table 3, below. The determination of whether an amino acid
residue substitution is similar, conservative, or highly
conservative is based exclusively on the side chain of the amino
acid residue and not the peptide backbone, which may be modified to
increase peptide stability, as discussed below.
TABLE-US-00011 TABLE 3 Classification of Amino Acid Substitutions
Highly Similar Conservative Conservative Amino Acid in Amino Acid
Amino Acid Amino Acid Subject Polypeptide Substitutions
Substitutions Substitutions Glycine (G) A, S, N A n/a Alanine (A)
S, G, T, V, C, P, Q S, G, T S Serine (S) T, A, N, G, Q T, A, N T, A
Threonine (T) S, A, V, N, M S, A, V, N S Cysteine (C) A, S, T, V, I
A n/a Proline (P) A, S, T, K A n/a Methionine (M) L, I, V, F L, I,
V L, I Valine (V) I, L, M, T, A I, L, M I Leucine (L) M, I, V, F,
T, A M, I, V, F M, I Isoleucine (I) V, L, M, F, T, C V, L, M, F V,
L, M Phenylalanine (F) W, L, M, I, V W, L n/a Tyrosine (Y) F, W, H,
L, I F, W F Tryptophan (W) F, L, V F n/a Asparagine (N) Q Q Q
Glutamine (Q) N N N Aspartic Acid (D) E E E Glutamic Acid (E) D D D
Histidine (H) R, K, O* R, K, O R, K, O Lysine (K) R, H, O R, H, O
R, H, O Arginine (R) K, H, O K, H, O K, H, O *"O" represents
Ornithine.
Compositions
[0049] The present disclosure provides compositions that include an
antimicrobial polypeptide as described herein. For example, the
antimicrobial polypeptide can be any of the polypeptides listed in
Table 2 or a fragment or variant thereof that retains antimicrobial
activity. In certain embodiments, the antimicrobial polypeptide
included in the compositions of the invention will be a synthetic
polypeptide (e.g., made by chemical synthesis and/or produced
recombinantly).
[0050] The compositions of the invention can include a single
antimicrobial polypeptide, or combinations of different
antimicrobial polypeptides. The compositions can be substantially
free of proteins and other polypeptides. As used herein, the term
"substantially free of proteins and other polypeptides" means that
less than 5% of the protein content of the composition is made up
of proteins and other polypeptides that are not an antimicrobial
polypeptide of the invention. A composition that is substantially
free of non-antimicrobial polypeptides of the invention can have
less than 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or less of
other non-antimicrobial polypeptides.
[0051] The compositions of the invention in certain embodiments
contain an antimicrobial polypeptide that is not naturally found in
a human or other mammal or animal.
[0052] The compositions of the invention can include at least 1 mg
(e.g., at least 5, 10, 20, 30, 40, 50, 75, 100, 150, 200, 250, 300,
400, 500, 600, 700, 800, 900, 1000 mg, or more) of antimicrobial
polypeptide. Thus, for example, the compositions can include an
amount of antimicrobial polypeptide equal to about 1 mg to about
1000 mg (e.g., about 5 mg to about 900 mg, about 5 mg to about 800
mg, about 5 mg to about 700 mg, about 5 mg to about 600 mg, about
10 mg to about 500 mg, about 10 mg to about 400 mg, about 10 mg to
about 300 mg, about 10 mg to about 250 mg, about 10 mg to about 200
mg, about 10 mg to about 150 mg, about 10 mg to about 100 mg, about
50 mg to about 500 mg, about 50 mg to about 400 mg, about 50 mg to
about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200
mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about
75 mg to about 500 mg, about 75 mg to about 400 mg, about 75 mg to
about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200
mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about
100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg
to about 300 mg, about 100 mg to about 250 mg, about 100 mg to
about 200 mg, or any other range containing two of the foregoing
endpoints).
[0053] The compositions of the invention can include a solution
that contains at least 1 mg/ml (e.g., at least 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg/ml
or more) of an antimicrobial polypeptide. Thus, for example, the
compositions can include a solution having an antimicrobial
polypeptide concentration of about 1 mg/ml to about 1000 mg/ml
(e.g., about 5 mg/ml to about 900 mg/ml, about 5 mg/ml to about 800
mg/ml, about 5 mg/ml to about 700 mg/ml, about 5 mg/ml to about 600
mg/ml, about 5 mg/ml to about 500 mg/ml, about 10 mg/ml to about
500 mg/ml, about 10 mg/ml to about 400 mg/ml, about 10 mg/ml to
about 300 mg/ml, about 10 mg/ml to about 250 mg/ml, about 10 mg/ml
to about 200 mg/ml, about 10 mg/ml to about 150 mg/ml, about 10
mg/ml to about 100 mg/ml, about 50 mg/ml to about 500 mg/ml, about
50 mg/ml to about 400 mg/ml, about 50 mg/ml to about 300 mg/ml,
about 50 mg/ml to about 250 mg/ml, about 50 mg/ml to about 200
mg/ml, about 50 mg/ml to about 150 mg/ml, about 50 mg/ml to about
100 mg/ml, about 75 mg/ml to about 500 mg/ml, about 75 mg/ml to
about 400 mg/ml, about 75 mg/ml to about 300 mg/ml, about 75 mg/ml
to about 250 mg/ml, about 75 mg/ml to about 200 mg/ml, about 75
mg/ml to about 150 mg/ml, about 75 mg/ml to about 100 mg/ml, about
100 mg/ml to about 500 mg/ml, about 100 mg/ml to about 400 mg/ml,
about 100 mg/ml to about 300 mg/ml, about 100 mg/ml to about 250
mg/ml, about 100 mg/ml to about 200 mg/ml, about 10 mg/ml to about
150 mg/ml, or any other range containing two of the foregoing
endpoints).
[0054] The compositions of the invention include pharmaceutical
compositions. Such pharmaceutical compositions can comprise one or
more antimicrobial polypeptides and a pharmaceutically acceptable
carrier. Pharmaceutical compositions can further include an active
ingredient other than an antimicrobial polypeptide of the
invention. The other active ingredient can be a
therapeutic/antimicrobial agent, such as a conventional antibiotic.
The conventional antibiotic can have antimicrobial properties or
other properties that the antimicrobial polypeptides of the
invention augment or are augmented by. In certain embodiments the
pharmaceutical composition includes a carrier, e.g., a carrier
protein such as serum albumin (e.g., HAS, BSA, etc.), which can be
purified or recombinantly produced. By mixing the antimicrobial
polypeptide(s) in the pharmaceutical composition with serum album,
the antimicrobial polypeptides can be effectively "loaded" onto the
serum albumin, allowing a greater amount of antimicrobial
polypeptide to be successfully delivered to a site of infection.
The pharmaceutical compositions of the present invention can be
formulated for oral administration, parenteral administration,
topical administration, or the like. Compositions formulated for
oral delivery can, for example, include an enteric coat, to ensure
that antimicrobial peptides contained therein reach the intestine
and beyond. Compositions formulated for topical delivery can be,
for example, suspended in a gel or cream or infused into a bandage,
to extend the duration of action of the antimicrobial peptides
contained therein. Alternatively, the antimicrobial peptides of the
invention can be coated on the surface of medical devices, such as
surgical instruments and indwelling medical devices (e.g.,
pacemakers, catheters, artificial joints, and the like), as a means
of preventing infection.
Methods
[0055] The antimicrobial polypeptides of the invention provide
powerful tools for treating or preventing a microbial infection in
a subject. Accordingly, the invention provides methods of
eliminating, reducing the number of, or significantly reducing the
replication of at least one microbial organism in a subject. The
subject can be any animal, such as a domesticated animal (e.g., a
horse, cow, pig, goat, sheep, rabbit, chicken, turkey, duck, etc.),
a pet (e.g., a dog, cat, rabbit, hamster, gerbil, bird, fish,
etc.), a lab animal (e.g., a mouse, rat, monkey, chimpanzee, owl,
fish, etc.), a zoo animal (e.g., a gorilla, orangutan, chimpanzee,
monkey, elephant, camel, zebra, boar, lion, tiger, giraffe, bear,
bird, etc.), a wild animal (e.g., a deer, wolf, mountain lion,
bird, etc.), or a human subject (e.g., a patient).
[0056] The antimicrobial polypeptide(s) can be administered at a
dose and frequency that depends on the type of animal, the size of
the animal, and the condition being treated. Typically, the
antimicrobial polypeptide is administered daily (or every other
day, or weekly), in an amount between about 1 mg and about 1000 mg
(e.g., about 5 mg to about 900 mg, about 5 mg to about 800 mg,
about 5 mg to about 700 mg, about 5 mg to about 600 mg, about 10 mg
to about 500 mg, about 10 mg to about 400 mg, about 10 mg to about
300 mg, about 10 mg to about 250 mg, about 10 mg to about 200 mg,
about 10 mg to about 150 mg, about 10 mg to about 100 mg, about 50
mg to about 500 mg, about 50 mg to about 400 mg, about 50 mg to
about 300 mg, about 50 mg to about 250 mg, about 50 mg to about 200
mg, about 50 mg to about 150 mg, about 50 mg to about 100 mg, about
75 mg to about 500 mg, about 75 mg to about 400 mg, about 75 mg to
about 300 mg, about 75 mg to about 250 mg, about 75 mg to about 200
mg, about 75 mg to about 150 mg, about 75 mg to about 100 mg, about
100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg
to about 300 mg, about 100 mg to about 250 mg, about 100 mg to
about 200 mg, or any other range containing two of the foregoing
endpoints). The daily dose can be administered once during the day,
or broken up into smaller doses that are taken at multiple time
points during the day. For a human (and other similarly-sized
mammals), a dose of 5 mg/kg every other day can be administered.
The antimicrobial polypeptide can be administered for a fixed
period of time (e.g., for 2-3 weeks), at intervals (e.g.,
administer polypeptide for 2-3 weeks, wait 2-3 weeks, then repeat
the cycle), or until such time as the microbial organism has been
eliminated or significantly reduced, the symptoms of the microbial
infection have been ameliorated, or the potential microbial
infection risk has been reduced or eliminated (e.g., a wound has
healed).
[0057] The administration of the antimicrobial polypeptides (or
pharmaceutical compositions comprising such polypeptides) in
conjunction with any of the foregoing methods can be performed
intravenously, intraperitoneally, parenterally, orthotopically,
subcutaneously, topically, nasally, orally, sublingually,
intraocularly, by means of an implantable depot, using
nanoparticle-based delivery systems, microneedle patch,
microspheres, beads, osmotic or mechanical pumps, and/or other
mechanical means.
[0058] In conjunction with any of the foregoing methods, the
antimicrobial polypeptides (or pharmaceutical compositions
comprising such polypeptides) can be administered in combination
with another drug, e.g., an antibiotic, antiviral, antifungal,
antiprotozoal, antimalarial, or a drug for treating a
non-infectious disease or other condition. In certain embodiments,
the other drug is one that can reduce a symptom of a
disease/microbial infection (e.g., to reduce or prevent a fever, to
treat or prevent nausea, etc.). In each case, the antimicrobial
polypeptide can be administered prior to, at the same time as, or
after the administration of the other drug.
EXAMPLES
[0059] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention, nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is average molecular
weight, temperature is in degrees Centigrade, and pressure is at or
near atmospheric.
Example 1: Activity Against Planktonic Gram-Negative and
Gram-Positive Bacteria
[0060] Peptides were tested against the following challenge
organisms by the M11-A8E CLSI standard for Antimicrobial
Susceptibility Testing of Anaerobic Bacteria: Enterococcus faecium
ATCC 700221; Enterobacter aerogens ATCC 13048; Staphylococcus
aureus MRSA ATCC 33591; Streptococcus pneumoniae ATCC 49619;
Pseudomonas aeruginosa ATCC 27853; Acinetobacter baumannii ATCC
17978D-5; Pseudomonas aeruginosa ATCC 19660; and Staphylococcus
epidermidis ATCC 51625. Sample dilutions ranged from initial sample
to 1:2048. Eleven (11) concentrations were tested in duplicate on a
96 well plate by MQA Laboratories. Results are shown in Table 4 as
Minimum bactericidal concentration (MBC), which is the
concentration of each peptide necessary to yield 99.9% lethality
for each of the eight challenge organisms. (The concentrations in
Table 4 are averages of the MBC across all bacteria tested in
.mu.M).
TABLE-US-00012 TABLE 4 Average MBC Peptide SEQ ID NO (.mu.M) RP438
17 5.3 RP444 23 7.5 RP441 20 8.8 RP445 24 10.4 RP443 22 11.3 RP442
21 15.2 RP440 19 18 RP439 18 18.4 RP435 14 19.6 RP437 16 27.4 RP436
15 40.9
Example 2: Activity Against Biofilm Bacteria
[0061] The Minimum Biofilm Eradication Concentration (MBEC) Assay
was used. MBEC values provide estimates on the concentration of an
antimicrobial product required to kill biofilm bacteria. The
Calgary Biofilm Device (CBD) plate was used to effect biofilm
formation on a lid containing 96 pegs. Bacterial cultures were
grown and diluted in Tryptic Soy Broth (TSB) to approximately
1.times.10.sup.7 CFU/mL before inoculation of the CBD plate, which
was then incubated for 24 hr at 35.degree. C. on a shaker at 125
rpm.
[0062] The peg lid containing biofilm was first rinsed in PBS to
remove planktonic cells prior to treatment with 2-fold serial
dilutions of test articles and control overnight at 35.degree. C.
The peg lid was rinsed in PBS twice before sonication in fresh
media to disrupt biofilm adhered to the pegs. The plate was then
incubated overnight to evaluate growth. Bacterial quantification
was performed by measuring absorbance at 650 nm (A650). By
definition, A650 reading of less than 0.1 indicates biofilm
eradication. Results are shown in Table 5.
TABLE-US-00013 TABLE 5 Average MBEC (.mu.M) Peptide RP438 RP442
RP443 RP444 (SEQ ID (SEQ ID (SEQ ID (SEQ ID Bacteria NO: 17) NO:
21) NO: 22) NO: 23) S. aureus MRSA (G+) 12.89 14.68 7.36 14.41 S.
epidermis (G+) 12.89 14.68 3.68 14.41 A. baumanii (G-) 6.45 1.87
3.68 3.68 P. aeruginosa (G-) 51.43 234.28 57.62 57.62
Example 3: Activity Against Biothreat Bacteria (B.
thailandensis)
[0063] In vitro activity of the Test Articles and comparator
antibiotic (ceftazidime) were tested as follows: in a sterile
96-well plate, 1.times.10.sup.5 CFU per well of bacteria were
incubated with serial dilutions of antibiotic (control) and peptide
in 10 mM phosphate buffer (3 h, 37.degree. C.). Bacterial survival
was determined by serial dilution at each peptide concentration in
sterile PBS. Dilutions were plated in triplicate on nutrient agar
and incubated at 37.degree. C. for 24 h; colonies were then counted
to determine survival. Bacterial survival was calculated by the
ratio of the number of colonies on each experimental plate to the
average number of colonies in the control plates lacking any
antimicrobial peptide. The antimicrobial peptide concentration
required to kill 50% of B. thailandensis (EC50) was determined by
graphing percent survival versus log of peptide concentration. EC50
was determined by fitting the data to a standard sigmoidal
dose-response curve. Each experiment was performed with three
replicates. Table 6 shows the EC50 results for each peptide tested
and for the antibiotic Ceftazidime.
TABLE-US-00014 TABLE 6 Peptide EC50 in .mu.M RP438 11.21 (SEQ ID
NO: 17) RP442 0.95 (SEQ ID NO: 21) RP443 55.63 (SEQ ID NO: 22)
RP444 14.77 (SEQ ID NO: 23) Ceftazidime 39.13
Example 4: Antibacterial and Antifungal Activity of Selected
Peptides (IC50 Values)
[0064] Measurement of antimicrobial and anti-fungal activity was
determined by a standard micrometer dilution method. Briefly, cells
were grown overnight in media specified for each strain, and were
diluted in the same media. Serial dilutions of the peptides were
added to microtiter plates in a volume of 50 ul, followed by the
addition of 50 ul of bacteria or fungi, 5.times.10.sup.5 CFU/ml.
Plates were incubated at 37 degrees for 24 hours and the Minimum
Inhibitory Concentrations (MICs) were determined as the lowest
peptide concentration that inhibited 50% of bacterial growth. Table
7 shows the IC50 in uM for each bacterial isolate tested and Table
8 shows the IC50 in uM for each fungus tested.
TABLE-US-00015 TABLE 7 Bacterial Cell Results IC50 (in .mu.M)
Peptide RP500 RP501 RP504 (SEQ ID (SEQ ID (SEQ ID Bacteria NO: 25)
NO: 26) NO: 27) A. baumanii Isolate 6043 7.6 7.3 21.7 Isolate 4838
7.6 29.0 43.3 E. coli Isolate 6571 3.8 7.3 10.8 Isolate 6572 3.8
7.3 10.8 E. cloaca Isolate 6053 7.6 14.5 10.8 Isolate 6054 7.6 14.5
21.7 Pseudomonas Xen5 0.5 14.5 10.8 Staphylococcus Xen36 3.8 14.5
10.8 Average 5.3 13.6 17.6
TABLE-US-00016 TABLE 8 Fungal Cell Results IC50 (in uM) Peptide
RP504 RP505 RP507 RP508 (SEQ (SEQ (SEQ (SEQ ID NO: ID NO: ID NO: ID
NO: Fungus 27) 28) 29) 30) C. albicans Isolate Y-326 43.3 17.5 79.9
-- Isolate Y- 43.3 8.8 40.0 38.3 6359 C. krusei Isolate Y- 2.7 17.5
20.0 38.3 27803 Isolate Y- 2.7 17.5 20.0 38.3 27825 C. tropicalis
Isolate Y- -- 8.8 40.0 76.6 48158 Isolate Y- -- 8.8 40.0 38.3 48166
Average 23.0 13.2 43.3 46.0
Example 5: Screening of Peptides for In Vitro Bactericidal
Activity
[0065] Bacteria tested included Burkholderia cepacia strain Toronto
(B.c.), Porphyromonas gingivalis strains A7436 and HG405,
Actinobacillus actinomycetemcomitans strain A7154 (A.a.),
Fusobacterium nucleatum strain 1594 (F.n.), Escherichia coli strain
(E.c.), Staphylococcus aureus ATCC strain 29213 (S.a.), and
Pseudomonas aeruginosa strain (P.a.). All bacteria were grown in
appropriate media under appropriate atmosphere to the early
exponential phase of growth. Media were inoculated with a dose of
bacteria to assure a minimum of five doublings before harvesting.
The cultures were washed twice in saline by centrifugation and
resuspended in saline at suitable concentration. In the initial
screening, all peptides were used at a final concentration of 10
.mu.M in saline with the target bacteria at 10.sup.6 CFU/ml as
estimated by optical density at 660 nm. Controls were treated with
an equal volume of saline. The suspensions were incubated at
37.degree. C. in ambient atmosphere and aliquots removed temporally
(0 to 2 hrs) for quantitative recovery of colony forming units.
This allowed determination of the kinetics of killing of the
individual peptides with the different bacterial strains. In
general, there was little or no loss in viability of the various
strains throughout the two-hour test period in the saline control.
There was, however, significant loss in viability (>1
log.sub.10) in the controls of both Fusobacterium nucleatum and
Actinobacillus actinomycetemcomitans within the time period, but
there was no detectable reduction until after 30 minutes. Killing
was considered significant if there was greater than a one-log
reduction in recoverable CFU in the peptide-treated vs. the
saline-treated control. Peptides that failed to kill at 10 .mu.M
were considered inactive. Any peptide that resulted in greater than
two logs reduction was titrated by either two-fold, five-fold or
ten-fold dilutions prior to testing with 10.sup.6 CFU/ml of the
target bacteria. The endpoint titration is determined as the last
concentration of peptide (in .mu.M) that gives greater than
two-logs reduction in recoverable CFU vs. the saline-treated
control ("Two-log Reduction Concentration"). This two-log reduction
concentration for SEQ ID NOs: 12 and 6 for each bacteria tested is
shown in Table 9 along with the average value across all bacteria
(last column).
TABLE-US-00017 TABLE 9 Two-log Reduction Concentration (in .mu.M)
Bacteria SEQ ID NO B.c. A7436 HG405 A.a. F.n. E.c. S.a. P.a. Ave.
12 (RP-433) 0.12 0.16 0.36 0.10 1.25 0.63 0.15 0.90 0.46 6 1.25
1.88 2.50 0.40 3.75 0.63 1.26 ND 1.67
Example 6: Killing of Antibiotic Resistant Bacteria
[0066] Staphylococcus aureus, Pseudomonas aeruginosa, and
Clostridium difficile were tested for their sensitivity to RP-439
and RP-442 (SEQ ID NOs: 18 and 21, respectively). These organisms
are associated with hospital-acquired infections. The experiments
were performed as described in Example 5. FIG. 2 shows results
taken at 1 hour time point after addition of the indicated peptide
or vancomycin. As is clear in FIG. 2, S. aureus was effectively
killed by both peptides at a level similar to vancomycin. However,
it was found that these peptides showed a broader range of efficacy
than this conventional antibiotic. Specifically, RP-439 and RP-442
were effective in killing both P. aeruginosa and C. difficile,
whereas vancomycin was not effective against these microbial
organisms. Similar results were obtained whether the bacteria were
exposed in single cell format (shown in FIG. 2) or in biofilm form
(as described in the previous Examples; biofilm data for RP-442
against S. aureus and P. aeruginosa shown above; biofilm data not
shown for C. difficile and peptide RP-439).
[0067] The results provided in the Examples demonstrate the
efficacy of the antimicrobial peptides of the invention in killing
a wide range of microbial organisms, including those that cause
medically important human infections.
Embodiments
[0068] The following non-limiting embodiments are provided to
illustrate aspects of the present invention.
[0069] 1. An antimicrobial composition comprising an antimicrobial
peptide, wherein the antimicrobial peptide comprises an amino acid
sequence having at least 60% sequence identity to any one of SEQ ID
NOs: 23, 25, 17, 12, 13, 27, 1 to 6, 8, 9, 14 to 16, 18 to 22, 24,
26, and 28 to 30.
[0070] 2. The antimicrobial composition of embodiment 1, wherein
the antimicrobial peptide is effective in killing at least one
bacterial, fungal, or protozoal organism.
[0071] 3. The antimicrobial composition of embodiment 2, wherein
the organism is a species classified in a genus selected from the
group consisting of: Acinetobacter, Actinobacillus, Burkholderia,
Candida, Clostridium, Enterobacter, Enterococcus, Escherichia,
Fusobacterium, Porphyromonas, Pseudomonas, Staphylococcus, and
Streptococcus. In some embodiments, the organism is resistant to
one or more conventional antibiotics (e.g., an MRSA organism).
[0072] 4. The antimicrobial composition of embodiment 3, wherein
the organism is selected from the group consisting of:
Acinetobacter baumannii, Actinobacillus actinomycetemcomitans,
Burkholderia cepacia, Burkholderia thailandensis, Candida albicans,
Candida krusei, Candida tropicalis, Clostridium difficile,
Enterobacter aerogens, Enterobacter cloaca, Enterococcus faecium,
Escherichia coli, Fusobacterium nucleatum, Porphyromonas
gingivalis, Pseudomonas aeruginosa, Pseudomonas aeruginosa,
Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus
aureus, Staphylococcus epidermidis, and Streptococcus
pneumoniae.
[0073] 5. The antimicrobial composition of any one of embodiments 2
to 4, wherein the antimicrobial peptide is effective in killing
microbes growing as a microbial biofilm.
[0074] 6. The antimicrobial composition of any preceding
embodiment, wherein the antimicrobial peptide comprises an
amphipathic region having a cationic surface that binds to the
membrane of a microbial pathogen.
[0075] 7. The antimicrobial composition of any preceding
embodiment, wherein the antimicrobial peptide comprises a
polyproline helix structure.
[0076] 8. The antimicrobial composition of any preceding
embodiment, wherein the antimicrobial peptide comprises a
hydrophobic tail region on the N-terminus, C-terminus, or both,
wherein the hydrophobic tail region has a sequence of from 4 to 10
hydrophobic amino acids.
[0077] 9. The antimicrobial composition of embodiment 8, wherein
the hydrophobic tail region has the amino acid sequence set forth
in SEQ ID NO: 4.
[0078] 10. The antimicrobial composition of any preceding
embodiment, wherein the antimicrobial peptide comprises two
amphipathic regions.
[0079] 11. The antimicrobial composition of embodiment 10, wherein
the two amphipathic regions are joined together by a linker.
[0080] 12. The antimicrobial composition of embodiment 11, wherein
the linker comprises a bubble region or a beta turn.
[0081] 13. The antimicrobial composition of any one of embodiments
10 to 12, wherein the two amphipathic regions form a dimer
structure.
[0082] 14. The antimicrobial composition of any one of embodiments
10 to 13, wherein the two amphipathic regions have the same amino
acid sequence.
[0083] 15. The antimicrobial composition of any one of embodiments
10 to 13, wherein the two amphipathic regions have different amino
acid sequences.
[0084] 16. The antimicrobial composition of any preceding
embodiment, wherein the antimicrobial peptide comprises an amino
acid sequence selected from the group consisting of: SEQ ID NOs:
23, 25, 17, 12, 13, 27, 1 to 6, 8, 9, 14 to 16, 18 to 22, 24, 26,
28 to 30; amino acid sequences having 5 or fewer amino acid
differences from any one of SEQ ID NOs: 23, 25, 17, 12, 13, 27, 1
to 6, 8, 9, 14 to 16, 18 to 22, 24, 26, 28 to 30; and homodimers or
heterodimers thereof linked by a linker.
[0085] 17. A pharmaceutical composition comprising the
antimicrobial composition of any one of embodiments 1 to 16 and a
pharmaceutically acceptable carrier.
[0086] 18. The pharmaceutical composition of embodiment 17, wherein
the composition is formulated for oral administration, parenteral
administration, or topical administration.
[0087] 19. The pharmaceutical composition of embodiment 18, wherein
the composition is formulated for oral administration and further
comprises an enteric coating.
[0088] 20. The pharmaceutical composition of embodiment 18, wherein
the composition is formulated for topical delivery in a form
selected from the group consisting of: a gel suspension, a cream,
microneedle, and infusion into a bandage or topical patch.
[0089] 21. The pharmaceutical composition of any one of embodiments
17 to 20, further comprising an additional bioactive agent.
[0090] 22. The pharmaceutical composition of embodiment 21, wherein
the additional bioactive agent is selected from the group
consisting of: an antimicrobial agent, an anti-inflammatory drug,
an anti-nausea drug, an anti-pain medication, and combinations
thereof.
[0091] 23. The pharmaceutical composition of embodiment 17, wherein
the composition is formulated to be coated on the surface of an
implantable medical device.
[0092] 24. The pharmaceutical composition of embodiment 23, wherein
the medical device is selected from the group consisting of:
surgical instruments and indwelling medical devices.
[0093] 25. A method of treating or preventing a microbial infection
in a subject in need thereof, the method comprising administering a
pharmaceutical composition according to any one of embodiments 17
to 24 to the subject.
[0094] 26. The method of embodiment 25, wherein the pharmaceutical
composition is administered to the subject orally, parenterally, or
topically.
[0095] 27. The method of embodiment 25, wherein the pharmaceutical
composition is administered to the subject by applying the
composition to a surface of a medical device prior to inserting the
medical device into the subject.
[0096] 28. The method of any one of embodiments 25 to 27, wherein
the subject is selected from the group consisting of: a human, a
domesticated animal, a farm animal, and a zoo animal.
[0097] It will also be recognized by those skilled in the art that,
while the invention has been described above in terms of preferred
embodiments, it is not limited thereto. Various features and
aspects of the above described invention may be used individually
or jointly. Further, although the invention has been described in
the context of its implementation in a particular environment, and
for particular applications those skilled in the art will recognize
that its usefulness is not limited thereto and that the present
invention can be beneficially utilized in any number of
environments and implementations. Accordingly, the claims set forth
below should be construed in view of the full breadth and spirit of
the invention as disclosed herein.
Sequence CWU 1
1
30118PRTArtificial Sequencesynthetic amino acid sequence 1Arg Val
Phe Lys Lys Ala Phe Arg Lys Phe Lys Lys Leu Phe Lys Arg 1 5 10 15
Ala Phe 218PRTArtificial Sequencesynthetic amino acid sequence 2Phe
Ala Arg Lys Phe Leu Lys Lys Phe Lys Arg Phe Ala Lys Lys Phe 1 5 10
15 Val Arg 318PRTArtificial Sequencesynthetic amino acid sequence
3Phe Lys Arg Lys Ile Lys Ala Lys Leu Arg Phe Lys Ala Lys Val Arg 1
5 10 15 Leu Lys 45PRTArtificial Sequencesynthetic amino acid
sequence 4Phe Ala Phe Ala Phe 1 5 523PRTArtificial
Sequencesynthetic amino acid sequence 5Phe Ala Phe Ala Phe Arg Val
Phe Lys Lys Ala Phe Arg Lys Phe Lys 1 5 10 15 Lys Leu Phe Lys Arg
Ala Phe 20 623PRTArtificial Sequencesynthetic amino acid sequence
6Phe Ala Arg Lys Phe Leu Lys Lys Phe Lys Arg Phe Ala Lys Lys Phe 1
5 10 15 Val Arg Phe Ala Phe Ala Phe 20 76PRTArtificial
Sequencesynthetic amino acid sequence 7Cys Leu Gly Arg Phe Cys 1 5
818PRTArtificial Sequencesynthetic amino acid sequence 8Lys Ile Arg
Ala Lys Leu Cys Leu Gly Arg Phe Cys Ile Arg Ala Lys 1 5 10 15 Leu
Arg 918PRTArtificial Sequencesynthetic amino acid sequence 9Lys Ile
Lys Ala Arg Leu Cys Leu Gly Lys Phe Cys Ile Lys Ala Arg 1 5 10 15
Leu Lys 106PRTArtificial Sequencesynthetic amino acid
sequenceMISC_FEATURE(1)..(1)Xaa can be any naturally occurring
amino acidMISC_FEATURE(6)..(6)Xaa can be any naturally occurring
amino acid 10Xaa Gly Pro Gly Arg Xaa 1 5 116PRTArtificial
Sequencesynthetic amino acid sequence 11Phe Gly Pro Gly Arg Phe 1 5
1250PRTArtificial Sequencesynthetic amino acid sequence 12Phe Ala
Phe Ala Phe Lys Ala Phe Lys Lys Ala Phe Lys Lys Phe Lys 1 5 10 15
Lys Ala Phe Lys Lys Ala Phe Gly Pro Gly Arg Phe Ala Lys Lys Phe 20
25 30 Ala Lys Lys Phe Lys Lys Phe Ala Lys Lys Phe Ala Lys Phe Ala
Phe 35 40 45 Ala Phe 50 1350PRTArtificial Sequencesynthetic amino
acid sequence 13Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala
Lys Lys Phe 1 5 10 15 Ala Lys Phe Ala Phe Ala Phe Gly Pro Gly Arg
Phe Ala Phe Ala Phe 20 25 30 Lys Ala Phe Lys Lys Ala Phe Lys Lys
Phe Lys Lys Ala Phe Lys Lys 35 40 45 Ala Phe 50 1419PRTArtificial
Sequencesynthetic amino acid sequence 14Met Gly Phe Lys Leu Arg Ala
Lys Ile Lys Val Arg Leu Arg Ala Lys 1 5 10 15 Ile Lys Leu
1512PRTArtificial Sequencesynthetic amino acid
sequenceMISC_FEATURE(3)..(3)Xaa is ornithineMISC_FEATURE(8)..(8)Xaa
is ornithine 15Cys Val Xaa Leu Phe Pro Val Xaa Leu Phe Pro Cys 1 5
10 1616PRTArtificial Sequencesynthetic amino acid sequence 16Cys
Lys Leu Arg Phe Arg Gly Pro Gly Arg Ile Lys Val Arg Leu Cys 1 5 10
15 1726PRTArtificial Sequencesynthetic amino acid sequence 17Cys
Pro Gly Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala 1 5 10
15 Lys Lys Phe Ala Lys Phe Ala Phe Ala Phe 20 25 1818PRTArtificial
Sequencesynthetic amino acid sequence 18Lys Ile Arg Ala Lys Leu Cys
Leu Gly Arg Phe Cys Ile Arg Ala Lys 1 5 10 15 Leu Arg
1926PRTArtificial Sequencesynthetic amino acid sequence 19Lys Lys
Lys Pro Lys Pro Pro Tyr Leu Pro Lys Pro Lys Pro Pro Pro 1 5 10 15
Phe Phe Pro Pro Lys Leu Pro Pro Lys Ile 20 25 2026PRTArtificial
Sequencesynthetic amino acid sequence 20Phe Ala Phe Ala Phe Lys Ala
Phe Lys Lys Ala Phe Lys Lys Phe Lys 1 5 10 15 Lys Ala Phe Lys Lys
Ala Phe Gly Pro Cys 20 25 2122PRTArtificial Sequencesynthetic amino
acid sequence 21Phe Ala Phe Ala Phe Ala Phe Lys Lys Ala Phe Lys Lys
Phe Lys Lys 1 5 10 15 Ala Phe Lys Lys Ala Phe 20 2223PRTArtificial
Sequencesynthetic amino acid sequenceMISC_FEATURE(6)..(6)Xaa is
ornithineMISC_FEATURE(9)..(10)Xaa is
ornithineMISC_FEATURE(13)..(14)Xaa is
ornithineMISC_FEATURE(16)..(17)Xaa is
ornithineMISC_FEATURE(20)..(21)Xaa is ornithine 22Phe Ala Phe Ala
Phe Xaa Ala Phe Xaa Xaa Ala Phe Xaa Xaa Phe Xaa 1 5 10 15 Xaa Ala
Phe Xaa Xaa Ala Phe 20 2323PRTArtificial Sequencesynthetic amino
acid sequenceMISC_FEATURE(3)..(4)Xaa is
ornithineMISC_FEATURE(7)..(8)Xaa is
ornithineMISC_FEATURE(10)..(11)Xaa is
ornithineMISC_FEATURE(14)..(15)Xaa is
ornithineMISC_FEATURE(18)..(18)Xaa is ornithine 23Phe Ala Xaa Xaa
Phe Ala Xaa Xaa Phe Xaa Xaa Phe Ala Xaa Xaa Phe 1 5 10 15 Ala Xaa
Phe Ala Phe Ala Phe 20 2422PRTArtificial Sequencesynthetic amino
acid sequence 24Phe Ala Lys Lys Phe Ala Lys Lys Phe Lys Lys Phe Ala
Lys Lys Phe 1 5 10 15 Ala Phe Ala Phe Ala Phe 20 2531PRTArtificial
Sequencesynthetic amino acid sequenceMISC_FEATURE(11)..(12)Xaa is
ornithineMISC_FEATURE(15)..(16)Xaa is
ornithineMISC_FEATURE(18)..(19)Xaa is
ornithineMISC_FEATURE(22)..(23)Xaa is
ornithineMISC_FEATURE(26)..(26)Xaa is ornithine 25Arg Leu Ala Arg
Ile Val Gly Gly Phe Ala Xaa Xaa Phe Ala Xaa Xaa 1 5 10 15 Phe Xaa
Xaa Phe Ala Xaa Xaa Phe Ala Xaa Phe Ala Phe Ala Phe 20 25 30
2633PRTArtificial Sequencesynthetic amino acid
sequenceMISC_FEATURE(13)..(14)Xaa is
ornithineMISC_FEATURE(17)..(18)Xaa is
ornithineMISC_FEATURE(20)..(21)Xaa is
ornithineMISC_FEATURE(24)..(25)Xaa is
ornithineMISC_FEATURE(28)..(28)Xaa is ornithine 26Cys Arg Leu Ala
Arg Ile Val Cys Gly Gly Phe Ala Xaa Xaa Phe Ala 1 5 10 15 Xaa Xaa
Phe Xaa Xaa Phe Ala Xaa Xaa Phe Ala Xaa Phe Ala Phe Ala 20 25 30
Phe 2724PRTArtificial Sequencesynthetic amino acid
sequenceMISC_FEATURE(2)..(2)Xaa is ornithineMISC_FEATURE(4)..(4)Xaa
is ornithineMISC_FEATURE(6)..(6)Xaa is
ornithineMISC_FEATURE(13)..(13)Xaa is
ornithineMISC_FEATURE(19)..(19)Xaa is
ornithineMISC_FEATURE(21)..(21)Xaa is
ornithineMISC_FEATURE(23)..(23)Xaa is ornithine 27Phe Xaa Ile Xaa
Ala Xaa Leu Gly Gly Cys Leu Gly Xaa Phe Cys Gly 1 5 10 15 Gly Ile
Xaa Ala Xaa Leu Xaa Phe 20 2826PRTArtificial Sequencesynthetic
amino acid sequenceMISC_FEATURE(1)..(1)Xaa is
ornithineMISC_FEATURE(3)..(3)Xaa is
ornithineMISC_FEATURE(11)..(11)Xaa is
ornithineMISC_FEATURE(13)..(16)Xaa is
ornithineMISC_FEATURE(18)..(18)Xaa is
ornithineMISC_FEATURE(20)..(22)Xaa is
ornithineMISC_FEATURE(26)..(26)Xaa is ornithine 28Xaa Leu Xaa Ser
Leu Leu Lys Thr Leu Ser Xaa Ala Xaa Xaa Xaa Xaa 1 5 10 15 Leu Xaa
Thr Xaa Xaa Xaa Ala Ile Ser Xaa 20 25 2927PRTArtificial
Sequencesynthetic amino acid sequenceMISC_FEATURE(7)..(9)Xaa is
ornithineMISC_FEATURE(12)..(12)Xaa is
ornithineMISC_FEATURE(14)..(14)Xaa is
ornithineMISC_FEATURE(16)..(16)Xaa is ornithine 29Ala Leu Trp Met
Thr Leu Xaa Xaa Xaa Val Leu Xaa Ala Xaa Ala Xaa 1 5 10 15 Ala Leu
Asn Ala Val Leu Val Gly Ala Asn Ala 20 25 3027PRTArtificial
Sequencesynthetic amino acid sequenceMISC_FEATURE(7)..(9)Xaa is
ornithineMISC_FEATURE(12)..(12)Xaa is
ornithineMISC_FEATURE(14)..(14)Xaa is
ornithineMISC_FEATURE(16)..(16)Xaa is ornithine 30Ala Phe Ala Phe
Thr Ala Xaa Xaa Xaa Phe Ala Xaa Phe Xaa Ala Xaa 1 5 10 15 Phe Ala
Asn Phe Ala Phe Ala Gly Phe Asn Ala 20 25
* * * * *